The DNA-protein interactions involved in gene regulation will be studied by utilizing the biotin (bio) operon of Escherichia coli as a model system. The relationships between promoter sequence and promoter function will be found by using recombinant DNA methods and by in vitro mutagenesis with chemically synthesized oligonucleotides. The interaction of repressor molecules with the bio operator will be analyzed in vitro by binding studies and in vivo by repressor titration. The long-term objective of the project is to understand the precise mechanisms of bacterial gene regulation. The data obtained may be applied to problems of gene evolution, bacterial metabolism, expression of synthetic genes and specific regulation of gene expression.